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ASTAXANTHIN FROM SHRIMP SHELL WASTE
Uma Nath Ushakumari*, Ravi Ramanujan

1. Dale View College of Pharmacy and Research Centre , Punalal Trivandrum
India, Research Scholar Karpagam University, Coimbatore, Tamil Nadu,
India.
2. Department of Pharmaceutical Chemistry, Sankaralingham Bhuvaneswari
College of Pharmacy, Sivakasi, Tamilnadu India.

ABSTRACT
The aim of the present study was to propose a simple and efficient method for the isolation of high-value pigment,
astaxanthin, from a low-value raw material, shrimp waste. Hexane was used as extraction solvent. The concentration
obtained was 43.44 g/g. Mobile phase for TLC was acetone: hexane in the ratio 3:7 (v/v). The sample was
collected from the processing plants, Cochin, Kerala, India. The shrimp waste was transported to the laboratory in a
sterile container filled with ice. The organism was identified with the styles marketed under the trade name red
ring. The wastes generated from the shrimp are cephalothorax, abdominal shell and tail portion. Adhering meat
from the cephalothorax was removed and the waste was washed under running water and dried under shade. They
were packed in polyethylene bags and stored at -20C until use. A second set of sample was stored wet, after
removing the adhering meat, was packed in polythene covers and stored at -20C.
Keywords: Astaxanthin , Aristeus alcocki, petroleum ether, acetone, 0.73% NaCl





INTRODUCTION
Astaxanthin (Figure1), unlike some
carotenoids, does not convert to Vitamin-A
(retinol) in the human body. Too much
Vitamin A is toxic for a human, but
astaxanthin is not. However, it is a powerful
Antioxidant; it is 10 times more capable
than other carotenoids. While astaxanthin is
a natural nutritional component, it can be
found as a food supplement. The supplement
is intended for human, animal, and
aquaculture consumption. The commercial
production of astaxanthin comes from both
natural and synthetic sources [1].


Figure 1: Astaxanthin

The U.S.Food and Drug Administration
(FDA) approved astaxanthin as a food
colouring (or colour additive) for specific
uses in animal and fish foods. The European
Union (actually European Commission)
considers it food dye within the E number
system
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Astaxanthin pronounced as(as-tuh-zan'-thin
) is a carotenoid. It belongs to a larger class
of phytochemicals known as terpenes. It is
classified as a xanthophyll, which means
"yellow leaves". Like many carotenoids, it is
a colorful, fat/oil-soluble pigment.
Astaxanthin can be found in microalgae,
yeast, salmon, trout, krill, shrimp, crayfish,
crustaceans, and the feathers of some birds.
Professor Basil Weedon was the first to map
the structures of astaxanthin. The study of
the pharmacological activity of astaxanthin
is vast and wide. The aim and scope of the
present research work is to study the anti
bacterial activity of astaxanthin [2].

MATERIALS AND METHODS

Preparation of shrimp waste

Sample: Shell waste from the deep sea
shrimp Aristeus alcocki
The sample was collected from the
processing plants, Cochin, Kerala, India.
The shrimp waste was transported to the
laboratory in a sterile container filled with
ice. The organism was identified with the
styles marketed under the trade name red
ring. The wastes generated from the shrimp
are cephalothorax, abdominal shell and tail
portion. Adhering meat from the
cephalothorax was removed and the waste
was washed under running water and dried
under shade. They were packed in
polyethylene bags and stored at -20C until
use. A second set of sample was stored wet,
after removing the adhering meat, was
packed in polythene covers and stored at -
20C.

1g of wet shrimp shell waste was ground
with using 10 ml of acetone. The extract was
filtered using Whatman filter paper. The
sample is repeatedly extracted and filtered
with the fresh solvent until get the colourless
filtrate (3 times). The pooled extract was
collected in a separated conical flask and
12.5 ml of petroleum ether (BP 40-60C)
and 9.4 ml of 0.73% NaCl were added. After
thorough mixing the epiphase was collected.
To the lower phase an equal amount of
water was added, mixed well and then the
epiphase was collected. The pooled
epiphase was kept in water bath at 60C for
the evaporation of petroleum ether.

Table : 1 Physiochemical Parameters For Extraction

PARAMETERS METHODS
ACETONE SOLVENT
PETROLEUM ETHER BP 40-60
0
C
SODIUM CHLORIDE 0.73%
EVAPORATION TEMPERATURE
FOR PETROLEUM ETHER
60C




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Quantification of Astaxanthin
The extracted astaxanthin is redissolved in 3 ml of hexane and read @470 nm.
AST (g/g)= A x D x 106
100 x G x d x E1cm
Where,
AST concentration in g/g
A is absorbance
D is volume of extract in hexane
106 is dilution multiple
G is weight of sample in g
D is the cuvette width(1 cm)
E is extinction coefficient 2100.
OD @470 nm is 3.041


AST (g/g)= 3.041 x 3 ml x 106
100 x 1cm x 1g x 2100
=43.44 g/g.


DETERMINATION OF PROXIMATE
COMPOSITION OF SHRIMP SHELL
WASTE

Moisture Content Determination [3]
Two grams of each of the sample was weighed
into dried weighed crucible. The samples was put
into a moisture extraction oven at 105C and
heated for 3h. The dried samples was put into
desiccators, allowed to cool and reweighed. The
process was reported until constant weight was
obtained. The difference in weight was calculated
as a percentage of the original sample.

Percentage moisture = W2 - W1 x
100
W2 - W3
1

Where
W1 =Initial weight of empty dish
W2 =Weight of dish +undried sample
W3 =Weight of dish +dried sample

Ash Content Determination ([3]Two
grams of each of the samples was weight
into crucible, heated in a moisture extraction
oven for 3h at 100C before being
transferred into a muffle furnace at 550C
until it turned white and free of carbon. The
sample was then removed from the furnace,
cooled in a desiccator to a room temperature
and reweighed immediately. The weight of
the residual ash was then calculated as

Ash Content Percentage Ash = Weight
of Ash x 100
Weight of
original of sample
1

Crude Protein Determination [3] The
micro kjeldahl method described by
A.O.A.C (1990) was used. Two grams of
each of the samples was mixed with 10ml of
concentrated H2SO4 in a heating tube. One
table of selenium catalyst was added to the
tube and mixture heated inside a fume
cupboard. The digest was transferred into
distilled water. Ten millimeter portion of the
digest mixed with equal volume of 45%
NaOH solution and poured into a kjeldahl
distillation apparatus. The mixture was
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distilled and the distillate collected into 4%
boric acid solution containing 3 drops of
methyl red indicator. A total of 50ml
distillate was collected and titrated as well.
The sample was duplicated and the average
value taken. The Nitrogen content was
calculated and multiplied with 6.25 to obtain
the crude protein content.

This is given as percentage Nitrogen
= (100 x N x 14 x VF) T
100 x Va

Where
N=Normality of the titrate (0.1N)
VF=Total volume of the digest=100ml
T=Titre Value
Va=Aliquot Volume distilled

Fat Content Determination [3] Two grams
of the sample was loosely wrapped with a
filter paper and put into the thimble which
was fitted to a clean round bottom flask,
which has been cleaned, dried and weighed.
The flask contained 120ml of petroleum
ether. The sample was heated with a heating
mantle and allowed to reflux for 5h. The
heating was then stopped and the thimbles
with the spent samples kept and later
weighed. The difference in weight was
received as mass of fat and is expressed in
percentage of the sample.

The percentage oil content is percentage fat
=W2 - W1 x 100
W3 1

Where
W1 =weight of the empty extraction flask
W2 =weight of the flask and oil extracted
W3 =weight of the sample

Chitin estimation
1 gm of dry sample was ground to abount 32
mm mesh and digested in 100 ml of 2%
NaOH at 100C for one hour. The digest
was filtered on a medium tared sintered
glass crucible to refilter the residue. The
digested residue was transfred to a beaker,
treated for 12 hours at room temperature
with 100ml of 5% HCl and refiltered on the
sintered glass crucible. The residue was
washed with hot distilled water until there
was a negative test for chloride. The crucible
was dried at 110C for 16 hours and
weighed.

The percentage chitin
=(W2 - W1)g x 100
W
Where
W1 =weight of the crucible
W2 =weight of the crucible and residue
W =weight of the sample in grams
(W2 - W1)g =weight of residue in grams





Table No 2 : Proximate Composition of Shrimp Shell Waste


SAMPLE COMPONENTS Fresh (mean SD) Dried (mean SD)
Moisture 71.6 0.2 15.5 0.2
Ash 11.3 1.0 28.4 0.5
Chitin 9.1 0.4 24.8 0.8
Lipid 1.0 0.4 2.1 0.1
Protein 5.1 0.6 30.0 0.7
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Identification of Astaxanthin in shrimp
shell waste extract by thin Layer
chromatography (TLC)

Analysis of different components in the
shrimp shell extract was done using thin
layer chromatography (TLC) based on the
method of Kobayashi and Sakamoto, 1999.
For this, a small volume of the extract was
spotted on silica gel plate and developed
using acetone: hexane 3:7 (v/v). The
separated bands were identified using
standard astaxanthin (Source: Green algae;
Manufacturer: Sigma Chemicals, USA) and
internationally accepted Rf values for
astaxanthin monoester and astaxanthin
diester. The different fractions, astaxanthin,
astaxanthin monoester, astaxanthin diester
were quantified by scraping out the
respective bands in TLC plate. The
astaxanthin present in the scraped out
sample was redissolved in 5 ml of hexane
and quantified as described earlier.



Figure 2: The Rf values of different carotenoids in the extract form Aristeus alcoki
(mean of three determination)






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Table 3: Rf value of Carotenoid

Carotenoid Rf value
Astaxanthin diester 0.78
Astaxanthin monoester 0.60
Astaxanthin 0.33


RESULTS AND DISCUSSION

The details of the astaxanthin estimated have
been discussed in table no 3. The Rf value of
astaxanthin diester was 0.78 and for
monoester it is 0.60. For astaxanthin it was
0.33.The standard astaxanthin was obtained
from Sigma Aldrich and was used for
comparison of TLC. The product obtained
was fine orange red powder. The quantity
was 43.44g/g.

REFERENCES

1)Naguib YM. Antioxidant activities of astaxanthin
and related carotenoids. J Agric Food Chem
2000 Apr;48(4):1150-4.

2)Fish Physiology And Biochemistry Volume 27,
Numbers 1-2/September 2002, Page No: 71-80.

3)AOAC Method 1990.